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Enrichment of Low-molecular-weight Thiol Compounds - Thiol-affinity Enrichment using Thio-tagTM Tip -

Ver. 1 (2015/9) 1. Introduction

Thiol-containing biomolecules, such as L-glutathione, homocysteine and various cysteine-containing proteins, have vital roles in cellular metabolism, maintaining protein structure, and regulation of intracellular oxidation status. Although many methods have been developed for the detection of thiol groups, few are amenable to use in the high-throughput enrichment of intact thiol-containing molecules in small volumes or in low weights of samples. In 2014, Prof. Koike's group (Hiroshima University) reported that a zinc(II) complex of a macrocyclic tetraamine (1,4,7,10-tetraazacyclododecane: cyclen) acts as a selective thiol-binding tag molecule (called “Thio-tagTM”) in an aqueous solution. Here, we introduce a simple and efficient protocol to enrich low-molecular-weight thiol compounds using a Thio-tagTM product under near-physiological conditions. This method is based on immobilized metal affinity chromatography (IMAC) using a micropipette tip (Thio-tagTM Tip) containing highly cross-linked agarose gel linked to the thiol-binding tag molecule.

2. Description of Thio-tagTM Tip

Thio-tagTM Tip provides an efficient procedure for separation of low-molecular-weight thiol compounds from biological samples at physiological pH. The procedure of the thiol-affinity enrichment requires a 1-mL syringe attached with a silicon-tube adapter and the aqueous buffers for the binding, washing, and elution processes. Thio-tagTM Tip containing zinc(II)-bound Thio-tagTM gel is supplied in a preservation solution of 20%(v/v) 2-propanol. The Thio-tagTM gel has no irritant effect on the skin. Store the tip in a refrigerator at ca. 4˚C. Under this condition, the product is stable for at least one year.

3. Warning and Limitations Thio-tagTM Tip is not for use in human diagnostic and therapeutic procedures. Do not use internally or externally in human or animals. It's used only for research. Care should be taken to avoid contact with Thio-tagTM gel. In the case of contact with skin or eyes wash immediately with water.

4. Advantages of Thio-tagTM Tip Method

# The total time for the thiol-affinity enrichment is less than 15 min. # The method requires no special apparatus and the procedure is simple. # Thio-tagTM Tip preferentially captures a thiol molecule (R-SH) by formation of a coordination bond between thiolate (R-S–) and zinc(II) ion at pH 7.8. # The thiolate anion bound to Thio-tagTM molecule is air-stable for a few days at room temperature. If using aqueous 0.1 M HCl as an elution buffer, the eluted thiol molecule is stable under air condition. # Oxidized thiol derivatives such as disulfide, sulfate, and S-nitroso compounds can all be eliminated in the flow-through and washing fractions.

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5. Principle of Thio-tagTM Tip Method

Binding Washing Elution

6. Solutions for Thiol-affinity Enrichment Sol. A: Preservation solution 20%(v/v) 2-propanol/water (100 mL) # 2-propanol 20 mL # distilled water for preparation of the 100 mL solution a proper quantity Sol. B: Binding buffer 0.10 mol/L HEPES–NaOH (pH 7.8, 100 mL) # HEPES (MW = 238, 10 mmol) 2.38 g # aqueous 10 M NaOH solution (ρ = 1.33 g/mL) 0.64 mL (0.85 g) # distilled water for preparation of the 100 mL solution a proper quantity Note: Check the pH of 7.8 ± 0.1 by using a pH meter. A 10x buffer solution can be used as a 10 mL solution containing the same solutes. Sol. C: Washing buffer 0.10 mol/L HEPES–NaOH + 0.10 mol/L CH3COONa (pH 7.8, 100 mL) # HEPES (MW = 238, 10 mmol) 2.38 g # CH3COONa (FW = 82, 10 mmol) 0.82 g # aqueous 10 M NaOH solution (ρ = 1.33 g/mL) 0.64 mL (0.85 g) # distilled water for preparation of the 100 mL solution a proper quantity Note: Check the pH of 7.8 ± 0.1 by using a pH meter. Sol. D: Elution buffer 1 # aqueous 0.10 M HCl (pH <2, 100 mL) Note: Another acidic aqueous solution, 1% (w/v) CF3COOH can be used similarly. Sol. E: Elution buffer 2 (= Reactivation buffer 1) 0.10 M EDTA–NaOH buffer (pH 7.0, ca.100 mL) # aqueous 0.10 M EDTA·2Na solution (a commercial product) 100 mL # aqueous 10 M NaOH solution (a commercial product) a proper quantity for adjustment of pH to 7.0 ± 0.1 by using a pH meter. Sol. F: Reactivation buffer 2 0.10 mol/L Bis-tris–CH3COOH + 10 mmol/L Zn(CH3COO)2 (pH 6.8, 100 mL) # Bis-tris (MW = 209, 10 mmol) 2.1 g

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# aqueous 0.10 mol/L Zn(CH3COO)2 solution 10 mL # distilled water 70 mL # aqueous 1.0 M CH3COOH solution for adjustment of pH a proper quantity to 6.8 ± 0.1 by using a pH meter. # distilled water for preparation of the 100 mL solution a proper quantity 7. Basic Protocol for Thiol-affinity Enrichment Preparation of Thio-tagTM Tip 1) Wash the outside of Thio-tagTM Tip and the inside space over the upper filter using distilled water to remove the preservation solution (Sol. A). 2) After addition of 100 µL of Sol. C into the tip from the top, attach a 1-mL syringe with a silicon-tube adapter to the tip. Thio-tagTM moiety is an acetate-bound species. 3) Dispense the liquid out with air by using the empty syringe. 4) Steps 2) & 3) are repeated once more. Note: Please check that Thio-tagTM gel in the space (ca. 30 µL) between the two filters is compressed on the lower filter.

Flow-through Fraction (FT) 5) Prepare a sample solution (50 ~ 100 µL in a microtube) using Sol. B or the 10x buffer solution. The sample solution pH should be in ranged between 7 and 8. 6) Draw the sample solution gently into the tip by using the 1-mL syringe. All the sample solution is passed through the Thio-tagTM gel and then the liquid is moved above the upper spherical filter. 7) All the liquid in the tip is gently dispensed into the microtube used for the sample preparation. 8) Steps 6) & 7) are repeated five times, and then the liquid is dispensed completely to obtain a flow-through fraction (FT) in the microtube.

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Washing Fractions (W1, W2, and W3) 9) After remove the syringe, 50 or 100 µL of Sol. C is added into the tip from the top. 10) Attach a 1-mL syringe to the tip and the washing buffer is gently pushed into the space between two filters. 11) The thiolate-bound Thio-tagTM gel is resuspended in the space by gently moving the syringe piston up and down a few times. 12) All the liquid in the tip is gently dispensed into a microtube. 13) This washing operation [steps from 9) to 12)] is repeated again and the resulting liquids are collected as washing fractions, W1 and W2. 14) Prepare a microtube containing 50 or 100 µL of distilled water. 15) The water is gently drawn into the tip and then all the liquid is moved above the upper spherical filter. 16) The liquid in the tip is gently dispensed into a microtube to obtain a final washing fraction (W3).

Elution Fractions (E1, E2, and E3) 17) To elute the thiolate-bound to Thio-tagTM gel, 50 or 100 µL of Sol. D (or Sol. E) is added into the tip from the top. 18) Attach a 1-mL syringe to the tip and the elution buffer is gently dispensed into a microtube. 19) The liquid in the microtube is again gently drawn and passed through the Thio-tagTM gel to the upper spherical filter. 20) The Thio-tagTM gel is resuspended by gently moving the syringe piston up and down a few times. 21) Keep the suspended state for 1 min for Sol. D (or 30 min for Sol. E). 22) The liquid is gently dispensed into the microtube to obtain the first elution fraction (E1). 23) If necessary, the steps from 17) to 22) are repeated to obtain additional elution fractions (E2 and E3). 24) As for mass spectrometric analysis, desalting of the thiol-enriched sample (e.g., Cys-containing peptide) can be conducted by a commonly used method using reversed-phase resin.

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Note: As for using Sol. E (neutral pH buffer), the thiol-release is caused by a slower reaction of demetallation of Thio-tagTM to produce Zn2+–EDTA complex and free thiol compounds. Thus, the suspending time with Sol. E (at neutral pH) is much longer than that using Sol. C. A used Thio-tagTM Tip can be reactivated by formation of the thiol-free zinc(II) complex (see next section 8). Note: Other elution solutions (50 or 100 µL) with a suspending time of ca. 1 min at room temperature: # Thiolate anion exchange method: 10 mM ditiothreitol in 0.10 M HEPES/NaOH buffer (pH 7.4) # Thio-tag exchange method: 10 mM Zn2+–cyclen/NaOH buffer (pH 7.4: called Thio-tagTM buffer) Note: A thiolate anion bound to a Zn2+–cyclen moiety is very stable under air condition at room temperature for a few days.

8. Reactivation of Thio-tagTM Tip

1) Dispense the liquid in a used tip with air by using an empty syringe. 2) To eliminate residual thiol molecules and zinc(II) ions from Thio-tagTM gel, 100 µL of aqueous 0.10 M EDTA solution (Sol. E: Reactivation buffer 1) is added into the tip from the top. 3) Attach a 1-mL syringe to the tip and the buffer is gently pushed into the space between two filters. 4) The gel is resuspended in the space by gently moving the syringe piston up and down a few times. 5) Keep the suspended state for over 60 min at room temperature. 6) Dispense the liquid with air by using the empty syringe. 7) Washed the gel by gentle injection of 1 mL of distilled water from the top using a 1-mL syringe. 8) To form the thiol-free Thio-tagTM Tip (active form), 100 µL of Sol. F (Reactivation buffer 2) is added into the tip from the top. 9) Attach a 1-mL syringe to the tip and the buffer is gently pushed into the space between two filters. 10) The Thio-tagTM gel is resuspended in the space by gently moving the syringe piston up and down a few times. 11) Keep the suspended state for over 60 min at room temperature and leave the solution in the tip. 12) Preserve the tip in a 50-mL bottle containing 5 mL of 20%(v/v) 2-propanol/water before use. Note: If a sample contains large-molecular-weight biomolecules such as protein or polysaccharides,

those molecules could partially remain in Thio-tagTM gel even after the washing step. In that case, the elution fraction would include some impurities derived from the macromolecules. As for the peptide analysis of a protein digest, ultrafiltration is recommended before the thiol-affinity enrichment.

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9. Selective Separation of N-Acetylcysteine

· Sample solution: 20 nmol each of N-acethyl-amino acids in 50 µL of Sol. B · Washing solution: Sol. C (50 µL x 2: W1 and W2); distilled water (50 µL: W3) · Elution solution: Sol. D (50 µL x 3: E1, E2, and E3)

The separation experiment was conducted according to the basic protocol except using Sol. C for preparation of the sample solution. The recoveries of N-acethyl amino acids are shown in Fig. 1. The quantity in the each fraction was analyzed by reversed phase HPLC. The recovery of N-acethylcysteine in the elution fractions was 93%. The other N-acethyl-amino acids were all eliminated in the FT and W1–W3 fractions.

Fig. 1. Recoveries of N-acetyl-amino acids in the each fraction.

10. Selective Separation of Reduced Form of Glutathione

· Sample solution: 20 nmol of SH-glutathione and 10 nmol of SS-glutathione in 50 µL of Sol. C · Washing solution: Sol. C (50 µL x 2: W1 and W2); distilled water (50 µL: W3) · Elution solution: Sol. D (50 µL x 3: E1, E2, and E3)

The separation experiment was conducted according to the basic protocol at room temperature except using Sol. C for preparation of the sample solution. The recoveries of reduced and oxidized species are shown in Fig. 2. The quantity in the each fraction was analyzed by reversed phase HPLC. Total recovery of reduced glutathione was 90% in the elution fractions. The SS-form of glutathione was eliminated in the FT and W1–W3 fractions.

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Fig. 2. Recoveries of SS and SH forms of glutathione in the each fraction. 11. Selective Separation of Cysteine-containing Peptide

· Sample solution: Tryptic digest of 5 nmol β-casein# + 6 nmol enolase cysteine peptide (ECP) in 50 µL of pH 7.4 HEPES buffer

· Washing solution: Sol. C (50 µL x 2: W1 and W2); distilled water (50 µL: W3) · Elution solution: Sol. D (50 µL x 3: E1, E2, and E3)

The separation experiment was conducted in reference to the basic protocol at room temperature and pH 7.4. The total time for the separation experiment was within 15 min. The separation result was analyzed by a reverse phase HPLC with a gradient mode. The cysteine-containing peptide was preferentially eluted in the E1 fraction at recovery of 74%. #The β-casein digest has no cysteine-containing peptide. A small amount of a monophosphorylated peptide * from β-casein was eluted in the E1 fraction. ECP: Ile-Gly-Leu-Asp-Cys-Ala-Ser-Ser-Glu-Phe-Phe-Lys

Fig. 3. Chromatographic result for a mixture of tryptic digest of β-casein and ECP. FT is flow-through fraction. E1 is the first elution fraction.

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Appendix: To reduce residual washing and elution fractions in a Thio-tagTM Tip, we recommend a use of a micro-centrifuge (ca. 20 sec) and 1.5-mL micro-tubes attached with a silicon adaptor. The adaptor can be prepared from a commercially available 2-mL pipette bulb.

References on Thio-tagTM Chemistry: ・Simple enrichment of thiol-containing biomolecules by using zinc(II)–cyclen-functionalized magnetic beads, Journal of Separation Scienece, 37, 1601-1609 (2014), H. Fujioka, M. Tsunehiro, M. Kawaguchi, Y. Kuramoto,

H. Kurosaki, Y. Hieda, E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike ・Role of Zinc(II) in β-Lactamase II: A Model Study with a Zinc(II)-Macrocyclic Tetraamine

(1,4,7,10-Tetraazacyclododecane, Cyclen) Complex, Journal of American Chemical Society, 116, 8443-8449 (1994), T. Koike, M. Takamura, and E. Kimura